In heart failure, the molecular regulation of intracellular and intercellular myocardial signaling pathways is often profoundly perturbed. Many signaling proteins in cardiac myocytes, including G proteins, G protein- coupled receptors, calcium-regulatory proteins, and nitric oxide synthase- are localized in sarcolemmal caveolae. Cardiac myocyte caveolae represent highly specialized invaginations of the sarcolemma, and form the T-tubular system that organizes and regulates sarcomere calcium delivery. Myocyte caveolae contain the protein caveolin-3, a transmembrane protein that serves a scaffold for the localization of many signaling proteins. During the initial funding period of this SCOR, we discovered that the endothelial isoform of nitric oxide synthase (eNOS) is expressed in cardiac myocytes and that its activity is regulated by its interactions with caveolin-3. The central hypothesis to be tested in these studies is that nitric oxide synthase and other key caveolae-targeted signaling proteins are aberrantly regulated in heart failure. In Aim 1, we will determine the composition the composition and regulation of cardiac myocyte caveolae in normal and failing hearts, and characterize the signaling proteins present in cardiac myocyte caveolae following receptor activation in vivo and in vitro. We will perform cellular imaging of caveolae-targeted signaling proteins using confocal laser microscopy, and identify the intracellular sites of NO synthesis in cardiac myocytes using the newly developed fluorescent dye, diaminofluorescein. Both eNOS and iNOS will be studied in this context, analysis of iNOS localization may provide new information on the role of NO in myocardial depression in systemic sepsis. These cellular imaging studies of caveolae constituents will be analyzed in the murine heart failure models being studied by other SCOR investigators. In Aim 2, we will conduct fluorescence resonance energy transfer experiments to explore interactions between cardiac myocyte-derived NO, caveolin and Ca++-binding regulatory proteins in T-tubules. In Aim 3, we will explore the role of caveolin-3 in regulation of NO-dependent signaling pathways in the myocardium; these studies may yield insights into the pathophysiology of cardiomyopathies associated with muscular dystrophy syndromes. In Aim 4, we will characterize the electrophysiological phenotype of eNOS/null mice using programmed electrical stimulation and drug infusions, as well as ambulatory EGG monitoring in eNOS/null mice, including heart rate variability analysis. Since we have shown that eNOS importantly modulates the autonomic control of myocyte beating rate in vitro, the in vivo studies in Aim 4 may provide new insights into the molecular mechanisms of sudden cardiac death in heart failure.